Antisense oligos or vectors inhibit MRNA processing, stability or translation without necessarily affecting gene transcription. The idea behind triplex formation is to inhibit gene transcription itself, thereby stopping MRNA production. There are several experimental models for forming triple helices which do not rely on Watson-Crick base pairing. Instead, in one model, triplet formation is based on A-A and G-G hydrogen bonding to the underlying Watson-Crick duplex. Hogan et al have made a triplex sequence indentical to a purine rich stretch of c-myc DNA 1 15 bp ups from the PI transcription start site (270 bp upstream from P2). In vitro, Hogan et al found this sequence, and not its Watson-Crick complement, to inhibit the transcription of the c-myc gene. Postel and coworkers have recently demonstrated that a positive transcription factor, PuF, binds to this very region of the c-myc gene and up-regulates c-myc transcription. Thus, forming a triplex at this site should down-regulate transcription of the gene. We have synthesized this sequence and its scrambled counterpart in both Po and Ps versions and have examined its effects on the growth and viability of HL60 cells, promyelocytic leukemia cans which overexpress c-myc. Both Po and Ps versions of the triplex oligo inhibit growth, although the Ps version is more effective at lower concentrations, as might be expected due to its increased stability. The scrambled sequence had no effect in either case. In addition, we have demonstrated that triplex oligos will hybridize in vitro under physiological conditions to plasmids containing the appropriate sequences. We have utilized stable transfection of CAT and luciferase constructs to work out the most suitable parameters for triplex action. Triplex formation should be a means of interfering with activity of specific transcription factors, including steroid receptors, cyclic AMP dependent kinases and retinoids, in intact cells.